环状RNAmmu_circ_0000021介导miR-143-3p靶向调控NPY在心肌缺血再灌注微循环障碍的作用及机制研究

The no-reflow phenomenon during myocardial ischemia-reperfusion (I/R) results in a higher incidence of adverse clinical outcomes. Studies have suggested that microvascular dysfunction is considered to be a pathophysiologic sign of the no-reflow phenomenon. Neuropeptide Y (NPY), a 36-amino acid peptide, is able to induce an increase in both cytosolic and nuclear calcium in cardiomyocytes. Recent research shows that NPY induced the vasoconstrictor effects on the coronary microcirculation. One recent study reported that the plasma NPY levels correlated with indices of reperfusion and coronary microvascular resistance in ST-segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PPCI). In the current study, candidate miRNA targets for NPY were screened by using a bioinformatics algorithm, NPY RNA has a putative miR-143-3p binding sequence in the 3’ untranslated region (3’-UTR). The circRNA/microRNA interaction was predicted with starbase software. For miR-143-3p, the potential interact circRNA include mmu_circ_0000021 and mmu_circ_0001098. In preliminary experiment, the expression levels of mmu_circ_0000021, miR-143-3p and NPY was significantly changed in response to hypoxia/reoxygenation in vitro and ischemia-reperfusion in-vivo. Moreover, NPY protein were down-regulated in neonatal mouse ventricular myocytes (NMVMs) transfected with miR-143-3p mimic, but up-regulated by miR-143-3p inhibitor compared with control. We also found a biotin-labelled specific probe for mmu_circ_0000021 could pull down miR-143-3p, as analysed by the northern blotting. Therefore, based on the recent researches, bioinformatics algorithm and preliminary experiment, we propose the hypothesis that mmu_circ_0000021 will increase intracellular calcium concentration and coronary microvascular resistance via suppressing miR-143-3p by targeting NPY, which involved in regulating microvascular dysfunction induced by myocardial ischemia-reperfusion injury. In the present study, firstly，we observe the expression levels of mmu_circ_0000021, miR-143-3p, NPY mRNA and protein in response to hypoxia/reoxygenation in vitro and ischemia-reperfusion in-vivo. Secondly, we set out to confirm that NPY was a direct target of miR-143-3p while mmu_circ_0000021 directly targeted miR-143-3p in cultures of neonatal mouse ventricular myocytes. Thirdly, we will confirm that mmu_circ_0000021 regulates intracellular calcium concentration through adjusting miR-143-3p by targeting NPY in an in-vitro hypoxia-reoxygenation model. We will also establish animal model of myocardial ischemia-reperfusion injury in adult male C57/BL6 mice to confirm that mmu_circ_0000021 involved in regulating microvascular dysfunction via adjusting miR-143-3p by targeting NPY. The result of the present study will provide new molecular target for the prevention and cure of no reflow.

心肌缺血/再灌注（I/R）后无再流现象导致严重的不良预后，其病理生理基础是微循环障碍。研究表明，神经肽Y(NPY)引起细胞内钙离子浓度增加，在冠脉微循环产生缩血管效应。我们预实验发现I/R后mmu_circ_0000021、miR-143-3p、NPY表达明显变化，且三者间可能存在靶向关系。因此我们推测：mmu_circ_0000021介导miR-143-3p靶向调控NPY，致细胞内钙离子浓度增加，加重微血管收缩，参与心肌I/R微循环障碍发生发展。拟进行以下研究，首先，在心肌细胞缺氧/复氧(H/R)及心肌I/R模型检测mmu_circ_0000021、miR-143-3p、NPY表达。其次，确证三者间靶向关系。最后，分别在H/R和I/R模型证实mmu_circ_0000021介导miR-143-3p调控NPY，影响细胞内钙离子浓度及心肌微循环障碍。本课题将为研究无再流的分子靶点提供新资料。

急性ST段抬高型心肌梗死再灌注治疗后的“无再流现象”导致了严重的不良临床预后，其病理生理基础是微循环障碍。NPY可能致心肌细胞内钙离子浓度增加，通过兴奋-收缩耦联效应，对冠脉微循环系统产生缩血管作用，参与心肌I/R微循环障碍的发生发展。我们通过生物信息学技术和靶基因预测软件TargetScan、miRanda对microRNA和小鼠NPY基因的靶向匹配关系进行预测，挑选软件交集及预测值高的靶点，发现miR-143-3p和小鼠NPY基因可能存在靶标关系。我们基于小鼠心肌缺血/再灌注模型做环状RNA去线性测序，筛选其差异基因并进行数据分析，并进一步采取starbase数据库预测环状RNA，将两者取其交集最终确定环状RNAmmu_circ_0000021作为本次研究的环状RNA。我们进一步以RT-qPCR进行验证，发现环状RNAmmu_circ_0000021在体内及体外实验中表达存在差异。并进一步证实miR-143-3p与NPY的靶向关系以及miR-143-3p与环状RNA可相互作用；证实mmu_circ_0000021介导miR-143-3p靶向NPY，通过影响细胞内钙离子浓度进而调控心肌细胞H/R损伤。在小鼠心肌缺血/再灌注模型中证实环状RNAmmu_circ_0000021介导miR-143-3p靶向NPY，影响心肌缺血/再灌注微循环障碍，改善心功能。本研究证实环状RNAmmu_circ_0000021可以作为“海绵体”吸附miR-143-3p，进一步调控NPY靶基因，致心肌细胞内钙离子浓度增加，而钙离子可通过兴奋-收缩耦联效应，诱导心肌微血管收缩，参与心肌I/R中微循环障碍的发生发展，加重无再流。